This invention relates to a novel method for cross-linking a protein using an enzyme. More particularly, it relates to a novel method for cross-linking protein using a multi-copper oxidase such as laccase or bilirubin oxidase.
Protein materials which are cross-linked to have a high molecular weight or gelled by the cross-linking method of the present invention can be used in the field of food processing such as of raw fish meat paste, kamaboko (fish cake), fish/livestock meat sausage, tofu (soy bean curd), noodles, confectionery/bread, food adhesives, sheet-like meat food, yogurt, jelly and cheese. In addition, they can also be used as novel protein-derived materials in a wide range of industries including cosmetics, raw materials of microcapsules and carriers of immobilized enzymes.
As enzymes having a possibility of increasing molecular weight of protein by cross-linking reaction, transglutaminase, lysyl oxidase, protein disulfide-isomerase, protein-disulfide reductase, sulfhydryl oxidase, lipoxygenase, polyphenol oxidase (tyrosinase) and peroxidase have been known (Matheis and Whitaker, J. Food Biochemistry, 11, 309-327, 1987).
Among the above-described enzymes having a possibility of increasing molecular weight of protein by cross-linking reaction, a method for cross-linking protein by transglutaminase is well known. It is also known that this method has been broadly used mainly in the field of food processing based on the discovery of an inexpensive microbial transglutaminase which does not require the presence of calcium for the reaction (JP-B-6-65280 (the term xe2x80x9cJP-Bxe2x80x9d as used herein means an xe2x80x9cexamined Japanese patent publicationxe2x80x9d), Agric. Biol. Chem., vol. 69, no. 10, pp. 1301-1308).
The protein cross-linking reaction by transglutaminase, however, has the following problem. That is, since transglutaminase is an enzyme which forms an intramolecular or intermolecular bridge structure of protein as a result of the acyl rearrangement reaction generated between the xcex3-carboxyl group of glutamine residue and the xcex5-amino group of lysine residue in a protein, some species of protein can hardly become the substrate for the enzyme due to insufficient glutamine residues or lysine residues. For example, albumin proteins cannot be used as the substrate for transglutaminase under their native state.
Thus, although a possibility of using several enzymes as the enzyme-aided protein cross-linking method has been indicated, most of them are not sufficiently useful in their supplying amounts, costs, easiness in the purification, and the like. Even if the microbial transglutaminase is used for the cross-linking method, its application is limited because of a problem that the reaction does not occur in some protein species. In consequence, great concern has been directed toward the development of a protein cross-linking method which uses other enzymes.
The inventor of the present invention has conducted extensive studies on the the possiblity of cross-linking reaction of protein using a multi-copper oxidase such as laccase, bilirubin oxidase, ascorbic acid oxidase and ceruloplasmin and, as a result, found that protein can be cross-linked by the use of these enzymes. Based on the finding of the novel method for cross-linking protein using a multi-copper oxidase such as laccase, bilirubin oxidase, ascorbic acid oxidase or ceruloplasmin, the present invention provides a protein cross-linking method which has a completely different reaction mechanism from that of transglutaminase and which can produce gelled protein having new physical properties and characteristics, with expanding the range of protein species to be treated which were limited in the known protein cross-linking method by transglutaminase.
The present inventors have made screening of a broad range of natural resources for finding a novel enzyme which can increase molecular weight of protein and makes it to gel by a cross-linking reaction and, as a result, found that certain enzymes classified as multi-copper oxidase can increase molecular weight of protein to make it gel by directly acting upon the protein, thus resulting in the accomplishment of the present invention.